1. Field of the Invention
This invention relates to methods of immunoanalysis and more particularly to methods of immunoanalysis using precipitates of immunocomplexes.
2. Description of the Prior Art
Immunoanalysis is used extensively in biochemistry and clinical chemistry to determine the concentration of proteins and other antigenic materials in biological fluids, such as blood plasma. Immunoanalysis relies on the ability of an antibody, raised against an antigen, to combine with the antigen to form an immunocomplex. Conventionally, a known amount of either the antigen or antibody is reacted with an unknown amount of the complementary immunoreagent, and some property of the resulting immunocomplex is measured in order to determine the amount of the unknown component which was present. The immunocomplex formed by the interaction of an antigen and antibody is often insoluble in the aqueous media in which the immunoanalysis is conducted. This property of the immunocomplex forms the basis of immunoanalysis using the "precipitin" reaction which has been used extensively in immunoanalysis. A detailed discussion of the precipitin reaction and its use in immunoanalysis is found in Tietz, N. W., Ed., Textbook of Clinical Chemistry, W. B. Saunders Co. Philadelphia, 1986, pp. 212-215.
In a properly conducted precipitin reaction the total amount of precipitate of immunocomplex formed is proportional to the amount of immunoreagent present over a range of concentration of immunoreagent. Accordingly, when the concentrations are within this suitable range the precipitate can be merely collected and weighed. However, this simple form of immunoassay using the precipitin reaction has a number of drawbacks. The precipitate of immunocomplex may be difficult to collect and weigh. More important, the precipitate will not form or will redissolve if one of the immunoreagents is present in great excess. Even if the proportions of the reagents are within a range suitable for quantitative immunoanalysis, the formation of the precipitate may be slow, and accordingly, it may be difficult to tell whether the reaction is quantitatively complete. In addition, simple gravimetric analysis is not amenable to automated analytical methods which are favored for clinical immunoassays today.
Accordingly, immunoanalytical methods using the precipitin reaction have resorted to other methods of measuring the amount of immunocomplex precipitate present in an analytical sample. In particular, methods have been developed which use the light-scattering properties of the immunocomplex precipitate to determine the amount of precipitate formed in the immunoreaction. However, the precipitin reaction still suffers from the problems associated with slow attainment of equilibrium, limited range of quantitative relation between precipitate formed and concentration of immunoreagents, and the formation of smaller amounts of precipitate if one of the reagents, e.g., the antigen, is in excess. Since it is more common to analyze for an antigen by reacting a sample containing an unknown amount of antigen with a known quantity of antibody, this problem is usually described as the antigen excess problem.
A nephelometric apparatus for performing immunoanalysis using the precipitin reaction wherein the amount of precipitate formed is measured by its light scattering property, together with a method of immunoanalysis using nephelometry to determine the amount of precipitate present in the sample at a given time, is disclosed in Anderson, U.S. Pat. No. 4,157,871, the entire specification of which is incorporated herein by reference. In Anderson's apparatus the electrical signal representing the intensity of scattered light is differentiated to derive a reaction rate, and the rate signal is again differentiated to derive a peak reaction rate which is found to be characteristic of the concentration of analytes in the sample solution. Anderson discloses that the peak rate of formation of the precipitate may be used in place of the total amount of precipitate formed as a measure of the amount of antigen in the sample. Anderson also discloses methods of manipulating the data derived from the scatter versus time signal to permit a determination of the condition of the sample and determine whether antigen is present in excess amount. However, Anderson teaches only measuring the scatter signal essentially up to the time at which it attains its peak rate. Anderson does not teach following the formation of the precipitate at times after it reaches its peak rate of formation.
Another method of determining whether the sample in a nephelometric precipitin immunoassay is in an antigen excess or antibody excess condition is disclosed by Sternberg, U.S. Pat. No. 4,204,837, the entire specification of which is incorporated herein by reference. Sternberg uses the peak rate of precipitate formation as well as the time interval from the beginning of the reaction to the time of peak rate to determine whether an additional amount of antigen or antibody should be added to the test sample to determine unambiguously whether the sample is in antigen excess or antibody excess.
However, the prior art does not teach monitoring the amount of precipitate formed beyond the achievement of the peak rate or for a fixed time interval which may coincide with the entire precipitin reaction from initiation to end point. Accordingly, useful data which can be used to derive a more accurate value for the antigen concentration and for determining whether a given sample is in a condition of antigen excess has been neglected.
Hence, a need has continued to exist for a method of immunoanalysis using the precipitin reaction which uses the information available from observing the course of the formation of the immunocomplex precipitate in order to determine the concentration of antigen and to determine whether a given sample is in a condition of antigen excess.